Following a meal, increased blood glucose levels stimulate insulin release from the pancreas to act throughout the body to lower blood glucose levels. Important sites of action of insulin on glucose metabolism include facilitation of glucose uptake into skeletal muscle and adipocytes, and an increase of glycogen storage in the liver. Skeletal muscle and adipocytes is responsible for insulin-mediated glucose uptake and utilization in the fed state, making them very important sites for glucose metabolism.
Diabetes comprises two distinct diseases, viz. type 1 (or insulin-dependent diabetes) and type 2 (insulin-independent diabetes), both of which involve the malfunction of glucose homeostasis. Type 2 diabetes affects more than 350 million people in the world and the number is rising rapidly. Complications of diabetes include severe cardiovascular problems, kidney failure, peripheral neuropathy, blindness and even loss of limbs and death in the later stages of the disease. Type 2 diabetes is characterized by insulin resistance in skeletal muscle and adipose tissue (fat), and at present there is no definitive treatment. Most treatments used today are focused on treating dysfunctional insulin signaling or inhibiting glucose output from the liver and many of those treatments have several drawbacks and side effects. There is thus a great interest in identifying novel insulin-independent ways to treat different form of metabolic orders connected with dysregulation of glucose uptake such as type 2 diabetes.
In type 2 diabetes the insulin-signaling pathway is blunted in peripheral tissues such as fat and skeletal muscle. Methods for treating type 2 diabetes typically include lifestyle changes, as well as the administration of insulin or oral medications to help the body with the glucose homeostasis. People with type 2 diabetes in the later stages of the disease develop “beta-cell failure” or the inability of the pancreas to release insulin in response to high blood glucose levels. In the later stages of the disease patients often require insulin injections, in combination with oral medications, to manage their diabetes. In type 2 diabetes the insulin-signaling pathway is blunted in peripheral tissues and most common drugs have side effects including the said down regulation or desensitization of the insulin pathway and/or the promotion of fat incorporation in fat, liver and skeletal muscle, furthermore increased stimulation of proliferation of certain cells and a higher risk of promoting cancer. There is thus a great interest in identifying novel ways to treat metabolic diseases including type 2 diabetes that do not include these side-effects.
The molecular understanding of the signaling pathway below the insulin receptor has been a very hard problem to solve and has been occupying a great number of researchers since the discovery of insulin. In short, control of glucose uptake by insulin involves activation of the insulin receptor (IR), insulin receptor substrate (IRS), phosphoinositide 3-kinase (PI3K) and thus stimulation of phosphatidylinositol (3,4,5)-triphosphate (PIP3), mammalian target of rapamycin also called mechanistic target of rapamycin (mTOR), Akt/PKB (Akt) and TBC1D4 (AS160), leading to translocation of glucose transporter 4 (GLUT4). Akt activation is considered necessary for GLUT4 translocation.
Akt has multiple actions and regulates cellular metabolism and survival. Akt can promote cell survival both directly and indirectly. Akt can promote proliferation and differentiation and has impact on the cell cycle and migration of cells. Akt can influence transcription and translation. Akt has been implicated in angiogenesis and tumor growth. Thus Akt promotes changes in cells and tissues that can lead to cancer and other pathophysiological effects, such as obesity and negative effects on insulin signaling. It would thus be desirable to be able to increase glucose uptake without stimulating Akt to circumvent these side-effects.
Another important protein involved in the insulin-signaling pathway is mTOR. mTOR is regulated by several upstream pathways involved in energy uptake of the cell. mTOR is a complex that exists in two complexes: mTOR complex-1 (mTORC1), which includes the protein raptor, and mTOR complex-2 (mTORC2), which includes the protein rictor. PI3K has a key function upstream of mTOR in the insulin pathway by generating polyphosphoinositides in the plasma membrane, which function as a docking site for Akt. Thereby Akt is brought close to its activating kinases; PDK-1, which phosphorylates Akt on Thr308, and mTORC2, which phosphorylates Akt on Ser473 (Rowland, Fazakerley & James 2011).
Insulin and catecholamines are released in the body in response to quite different stimuli. Whereas insulin is released in response to the rise in blood sugar levels after a meal, epinephrine (also referred to as adrenaline) and norepinephrine (also referred to as noradrenaline) are released due to various internal and external stimuli, such as exercise, emotions and stress but also homeostatic tissue regulation. Insulin is an anabolic hormone that stimulates many processes involved in growth including glucose uptake, glycogen and triglyceride formation whereas catecholamines are mainly catabolic. Although insulin and catecholamines normally have antagonistic effects, it has been shown previously that they have similar actions in skeletal muscle on glucose uptake (Nevzorova et al. 2006). It is likely that catecholamines stimulate glucose uptake via adrenergic receptors to supply muscle cells with an energy substrate. Thus, it is likely that in mammals, including humans, the adrenergic and insulin systems can work independently to provide for the energy need of skeletal muscle during different situations. Since insulin stimulates many anabolic processes including a number of unwanted side effects it would be beneficial to be able to stimulate glucose uptake through the newly found adrenergic signaling pathway, which is catabolic and does not include many of the unwanted processes.
It is known in the field of the art that adrenergic receptors are prototypic models for the study of G protein-coupled receptors (GPCRs) and their signaling (Santulli, Iaccarino 2013, Drake, Shenoy & Lefkowitz 2006). There are three different classes of ARs, with distinct expression patterns and pharmacological profiles: α1-, α2- and β-ARs. The α1-Ars comprise the α1A, α1B and α1D while α2-ARs are divided into α2A, α2B and α2C. The β-ARs are also divided into the subtypes β1, β2, and β3, of which β2-AR is the major isoform in skeletal muscle cells (Watson-Wright, Wilkinson 1986, Liggett, Shah & Cryer 1988). Adrenergic receptors are G protein coupled and signal through second messengers such as cAMP and phospholipase C and are thus suited as prototypical models for most classes of GPCRs.
Glucose uptake in cells is mainly considered to be through facilitative glucose transporters (GLUT). GLUTs are transporter proteins mediating transport of glucose and/or fructose over the plasma membrane down the concentration gradient. There are fourteen known members of the GLUT family, named GLUT1-14, divided into three classes (Class I, Class II and Class III) dependent on their substrate specificity and tissue expression. GLUT1 and GLUT4 are the most intensively studied iso forms and, together with GLUT2 and GLUT3, belong to Class I which mainly transports glucose (in contrast to Class II that also transports fructose). GLUT1 is ubiquitously expressed and is responsible for basal glucose transport. GLUT4 is only expressed in peripheral tissues such as skeletal muscle, cardiac muscle and adipose tissues. GLUT4 has also been reported to be expressed in e.g. brain, kidney, and liver. GLUT4 is the major iso form involved in insulin stimulated glucose uptake.
To treat a condition involving a dysregulation of glucose homeostasis or glucose uptake in a mammal, it would be very advantageous to be able to activate certain GLUTs. For example for diseases such as type 2 diabetes it is vital to activate GLUT4 translocation to the plasma membrane and thus glucose uptake. Regulation of GLUT1 translocation or intrinsic activity has been suggested to occur in several tissues including erythrocytes depending on ATP-levels (Hebert, Carruthers 1986). It has also been indicated in HEK-cells (Palmada et al. 2006), 3T3-L1 (Harrison et al. 1992) and clone-9 cells (Barnes et al. 2002). Impaired GLUT translocation, of in particular GLUTS, has been reported as involved in both male and female infertility (Gawlik et al. 2008, Carayannopoulos et al. 2000). The mechanism whereby insulin signaling increases glucose uptake is mainly via GLUT4-translocation from intracellular storage to the plasma membrane (Rodnick et al. 1992). After longer insulin stimulation also GLUT1-content is increased due to increased transcription (Taha et al. 1995). Glucose uptake in type 2 diabetes is associated with defects in PI3K activity, insulin receptor tyrosine, IRS and Akt phosphorylation, resulting in impairment of GLUT4 translocation to the plasma membrane. Impaired GLUT translocation also plays a role in muscle wasting. Furthermore, GLUT translocation plays a role in feeding behavior. Mice lacking GLUT4 develop problems with lipid and glucose homeostasis leading to changes in feeding behavior. Decreased concentrations of GLUT1 and GLUT3 have also been shown in the brains of patients with Alzheimer's disease (Simpson et al. 2008). Also in a review article of Shah K, et al. (Shah, Desilva & Abbruscato 2012) the role of glucose transporters in brain disease, diabetes and Alzheimer's disease is discussed.